AIM: To investigate the presence of human papillomavirus (HPV) DNA along with the integration, the quantification and the expression of the HPV16 in colorectal cancers.

METHODS: A prospective series of colorectal tumors were genotyped for HPV DNA. The clinical and pathological variables of the HPV-positive tumors were compared to those of HPV-negative samples. The integration status of HPV16 was evaluated by calculating E2/E6 ng ratios. HPV16-positive tumors were also evaluated for (1) E2, E4, E5, E6 and E7 viral gene ng quantification; (2) relative quantification compared to W12 cells; and (3) viral E2, E4, E5, E6 and E7 mRNA transcripts by real-time polymerase chain reaction.

RESULTS: HPV infection was detected in 16.9% of all tumors examined, and HPV16 was the most frequent type detected (63.6% of positive tissues). Notably, the clinical and pathological features of HPV-positive colorectal cancers were not significantly different than those of HPV-negative cancers (χ² and t-test for all clinical and pathological features of HPV-positive vs HPV-negative colorectal cancers: p ns). HPV16 DNA was present exclusively in episomal form, and the HPV16 E2, E4, E5, E6 and E7 genes were detected in trace nanogram quantities. Furthermore, the HPV16 genes ranged from 10^-3 to 10^-9 compared to W12 cells at an episomal stage. Although the extractions were validated by housekeeping gene expression, all the HPV16 positive tissues were transcriptionally inactive for the E2, E4, E5, E6 and E7 mRNAs.

CONCLUSION: Based on our results, HPV is unlikely involved in colorectal carcinogenesis.

Colorectal carcinoma has been classified as the fourth most frequently diagnosed cancer in Italy and in the United States[1,2].

Recently, a possible correlation between human papillomavirus (HPV) infection and colorectal cancer has been suggested, and several authors detected HPV DNA in cancerous tissues by different techniques[3]. As we recently highlighted, HPV infection rates are significantly different between tumors and controls/tumor-adjacent tissue when HPV DNA is detected, with the most prevalent type reported as HPV16[3]. Similar results have been validated by recent meta-analysis[4].

The literature lacks studies utilizing clinically validated methodologies for HPV genotyping, as well as studies investigating the molecular basis of HPV-associated colorectal carcinogenesis. Thus, a potential role for HPV in colorectal-related carcinogenesis requires further investigation.

The natural history of a high-risk HPV infection in the target epithelium (e.g., the cervix) is a well-described process that involves virus penetration in the basal layer through micro-abrasions and its subsequent replication in episomal form, leading to the expression of six non-structural (E1-E2, E4-E7) and two structural viral proteins (L1- L2)[5-7].

E5, E6 and E7 are viral onco-proteins that cause immortalization and transformation; E6 and E7 inactivate p53 and Rb, respectively[8]. HPV16 E5 also has transforming potential, leading to increased EGFR recycling and signaling[9].

The progression of a persistent and untreated lesion is associated with HPV DNA integration into the host genome, with subsequent disruption of the E2 gene and up-regulation of the E6 and E7 proteins[5,6].

HPV integration has been identified as a critical step in cancer promotion and progression[10]; however, in vivo studies have shown that an integrated, episomal or mixed HPV genome could be detected in pre-cancerous lesions in addition to invasive cancers[11].

Moreover, persistent and progressive HPV infection has been associated with increased viral load[12].

In summary, there are three major issues that scientists face in studying HPV-related carcinogenesis, including: (1) the expression of viral onco-proteins; (2) the integration pattern; and (3) the viral load of a productive/progressive infection.

On the basis of this background, we aimed to translate the molecular basis of HPV-related carcinogenesis in vivo (particularly HPV16), to understand the possible implications of the virus in colorectal carcinogenesis, in relation to: (1) the detection of HPV DNA in a prospective series of colorectal cancer tissues by a clinical validated methodology; (2) HPV16 integration status; (3) the quantification of HPV16; and (4) viral transcript expression.

HPV is no longer associated exclusively with cervical cancer[15,16]. Recent studies have detected HPV DNA in the tumoral mucosae of different epithelia[3,17,18].

Burnett-Hartman analyzed the correlation between HPV and colorectal cancer using the Bradford criteria[19] and highlighted evidence for the correlation by analogy, biological plausibility, strength of association, but only weakly provided evidence in consistency, specificity and coherence.

We recently reviewed this association by pooling patients from different studies: the HPV mean detection rate within colorectal carcinomas was 41.7% (range: 0%-84%), compared to a mean detection rate of 32.8% in adjacent colonic mucosae, and 5.8% in disease-free controls (P = 0.001)[20-22]. Moreover, HPV DNA has been reported to be statistically associated with colonic polyp mucosae[3]; conversely, two recent studies provided contrasting results[23,24].

Consistent with the findings of our previous review, Baandrup et al[4] recently reported an HPV prevalence of 36.8% in colorectal adenocarcinomas with an OR of 6.0 (95%CI: 2.0-17.9) for the association between HPV and colorectal cancer.

In our study, we documented an HPV DNA infection in 16.9% of the cancers analyzed. To our knowledge, this is the first study to use a clinically validated methodology. Conversely, the vast majority of past reports employed obsolete methods or used double amplification protocols[3]. According to Burnett-Hartman, prior studies are likely the result of contamination from the anal canal or clinical processing[23].

Despite this, HPV16 was the most frequently identified HPV type in tissues we examined (63.6%), in accordance with previous studies[20-22].

In this study, the presence of HPV showed no difference in distribution of the virus through the colon, as previously reported by other authors[20-22]. The prevalence of right colon localization in our samples may be explained by the fact that we excluded patients with rectal cancers that had previously undergone neo-adjuvant chemo-radiotherapy (which is currently the gold standard treatment for rectal cancers clinically staged T3 or N⁺), because of the radio-sensitivity related to HPV infection that could bias our results[13].

We thus compared the features of HPV-positive vs HPV-negative cancers, and the sub-groups appeared homogeneous for all clinical/pathological variables observed, in contrast to previous reports by Pérez et al[22] and Cheng et al[25], which highlighted a correlation between virus infection and tumor stage or grading.

Nevertheless, the main objective of our study was to translate the molecular basis of HPV-related carcinogenesis in vivo. Bodaghi was the only author who previously investigated HPV genome integration in colorectal tumors, detecting integrated virus quite frequently in the 31 tumor tissues investigated[21]. Of note, we investigated the entire HPV16 genome, providing an examination of the full viral structure. Indeed, in 57.1% of HPV16-positive tumors, we documented the entire viral genome; in the remaining cases, we detected only viral fragments.

To determine the amount of HPV16 DNA in positive tissues, we generated a relative quantification of the genes comparing to the episomal W12 cells. We detected very low quantities of HPV16 in all positive tissues.

Notably, the reported HPV prevalence of 16.9% in the colorectal cancers we examined is much lower than that of other HPV-associated cancers, as the prevalence of HPV in cervical cancer has been reported to be approximately 96%[26], and approximately 30%-44% in oropharyngeal cancers[27].

Chaturvedi et al[27] recently investigated a large series of oropharyngeal carcinomas using the Inno-Lipa technique; they determined that the HPV prevalence in oropharyngeal tumors was 44.1% (38.8% for HPV16). Among the 102 Inno-LiPA HPV16-positive tissues, 77.5% were positive based on viral load criterion, with a median of 22.1 HPV16 copies/cell, and 84.5% were positive by viral oncogene expression criterion, with a median of 152.9 transcripts/1000 RPLP0 equivalents.

Similar findings were reported in the investigation of HPV16 in cervical cancers; notably, Häfner et al[11] reported viral integration as a key event in malignant lesions: they found integrated HPV16 virus in 51% of cervical cancers tested by APOT assay. Conversely, just 11% of CIN lesions contained HPV16 integration. Furthermore, viral transcript levels were broadly distributed, though they had similar median values irrespective of histo-pathological grading and physical state of the viral genome both in CINs and cancers.

Indeed, we focused this study on viral transcript detection. Although all of the samples’ integrity was validated by housekeeping mRNA expression, all of the tissues were negative for HPV16 mRNAs. Conversely, Chen previously reported E6 protein detection by immunohistochemistry in eight out of eleven HPV16-positive colorectal tissues[28]. Our negative results could be explained by the very low virus quantities in positive tissues or by different transcriptional patterns of the virus in a different target epithelium that could impair viral protein replication and expression (simple cuboidal in colorectum vs squamous stratified in the cervix).

In conclusion, we were able to detect HPV DNA in colorectal cancer tissues; however, the virus was episomal, detectable in barely traceable quantities and transcriptionally inactive. This could be related to the very low amounts of viruses or to a different target epithelium that could bias a productive infection or the transcriptional patterns of the HPV. According to our results, it is unlikely that HPV contributes to colorectal carcinogenesis.

The authors thank Venuti A and Paolini F (Laboratory of Virology, Regina Elena Cancer Institute of Rome, Italy) for the scientific discussions and advice. Moreover, the authors thank Mr. Simone De Michelis for the technical help in the preparation of figures.